Highly sensitive detection method of amino acid derivative

ABSTRACT

To easily detect an amino acid derivative with high sensitivity in carrying out amino acid sequence analysis of a protein from an amino terminal, 2-anilino-5-thiazolinone amino acid derivative is reacted with a volatile primary amine represented by the general formula X-NH 2 , wherein X denotes a hydrocarbon containing a halogen to form a phenylthiocarbamyl amino acid derivative, and the phenylthiocarbamyl amino acid derivative is detected by using a gas chromatograph provided with an ECD. Accordingly, amino acid sequencing may be performed with high sensitivity without the need to use harmful radioisotope labeled or fluorescent amino compounds.

TECHNOLOGICAL FIELD

The present invention relates to an analysis method of amino acidsequence from an amino (N) terminal of protein.

BACKGROUND TECHNOLOGY

As shown in FIG. 2, for identification of an amino acid derivative inthe last stage of Edman degradation, which is a sequence analysis methodof a protein from the N terminal, protein is conventionally reacted withphenylisothiocyanate (PITC) to obtain phenylthiocarbamyl (PTC) protein,which is treated with an acid to produce 2-amino-5-thiazolinone (ATZ)amino acid derivative, which is further treated with an acid to formphenylthiohydantoin (PTH) amino acid derivative, which is detected byultraviolet absorption spectroscopy as a known analysis method (P.Edman, Acta Chem. Scand. 10, 761 (1956)).

Further, another method is disclosed in Japanese Patent ApplicationLaid-Open No. 61-264264, and shown herein in FIG. 3, in which an ATZamino acid derivative is reacted with an amino compound labeled with aradioactiveisotope to form a PTC amino acid derivative, which isseparated by thin-layer chromatography for detection.

Moreover, still another method is disclosed in Japanese PatentApplication Laid-Open No. 63-196858 and shown in FIG. 4 herein in whichan ATZ amino acid derivative is reacted with a fluorescent aminocompound to form a PTC amino acid derivative, which is separated by highspeed liquid chromatography for detection.

With regard to the conventional method of detecting the PTH amino acidderivative by ultraviolet absorption spectroscopy, while the detectingmeans is rather simple, this sequencing method is not suitable for thehighly sensitive analysis which is recently demanded for treating atrace amount of protein or very trace amount of peptide.

Further, with regard to the developed highly sensitive analysis methodutilizing the amino compound labeled with the radioactiveisotope, thishas rather limited application in view of ill effects to the environmentand, in particular, to the human body.

Moreover, with regard to the other developed highly sensitive analysismethod utilizing the fluorescent amino compound, it is necessary to adda large quantity of a fluorescent reagent for maintaining a reactionrate when treating a trace amount of sample in such a liquid phasereaction. Further, it is known that the reaction yield rate decreases asa density of the sample, i.e., the ATZ derivative of protein or peptidedecreases even though a large quantity of the reactive reagent is added.In such a case, the excess fluorescent amino compound which remainsafter the reaction with the ATZ amino acid derivative disturbsquantitative analysis of the product. Further, since the fluorescentcompound is nonvolatile, it is complicated to remove the same after thereaction with the ATZ amino acid derivative.

In view of this, an object of the present invention is to provide a newmethod of promoting a high yield rate reaction to readily obtain the PTCamino acid derivative for highly sensitive detection.

SUMMARY OF THE INVENTION

For solving the above noted drawback, according to the presentinvention, in order to analyze the amino acid sequence from an Nterminal of a protein, an ATZ amino acid derivative is reacted with avolatile primary amine represented by a general formula X-NH₂ (X denotesa hydrocarbon containing a halogen) to thereby produce a PTC amino acidderivative. Then, the PTC amino acid derivative is detected by a gaschromatograph provided with an electron capture detector (ECD).

By this means, the high yield rate reaction is promoted to readilydetect the PTC amino acid derivative with a high degree of sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a step diagram showing the inventive analysis method. FIG. 2shows one conventional analysis method of detection by ultravioletabsorption spectroscopy. FIG. 3 shows another conventional analysismethod utilizing an amino compound labeled with a radioactiveisotope.FIG. 4 shows a still another conventional analysis method utilizing afluorescent amino compound. FIG. 5 shows a gas chromatograph fordetecting a PTC phenylalanine derivative, where (a), (b) and (c) showanalysis results of the PTC phenylalanine derivative in the amounts of 5pmol, 500 fmol and 50 fmol, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The detailed description is given for the invention in conjunction withthe attached drawings. The embodiment 1 indicates that the detectionsensitivity of an amino acid derivative is significantly high accordingto the invention.

First, FIG. 1 shows steps of the inventive method. An amino compound ofhydrocarbon containing a halogen is applied to an ATZ amino acidderivative obtained by a process based on Edman degradation to form aPTC amino acid derivative. Next, this PTC amino acid derivative isidentified by a gas chromatograph provided with an ECD.

The PTC phenylalanine amino acid derivative is prepared by the followingsteps. First, a dipeptide of Phe-Gly is subjected to Edman degradationto obtain an ATZ phenylalanine derivative. This process of the Edmandegradation is known, and is therefore not described in detail here.Then, the ATZ phenylalanine derivative is filled in a test tube of φ4×40mm, and is dried. This test tube is then placed into another test tubeof φ13×100 mm provisionally filled with 100 μl of2,2,2-trifluoroethylamine (TFEA). The outer test tube is vacuum-sealedto carry out a reaction for 20 minutes at 80° C. After opening the testtube, an excess of TFEA is removed by blow of nitrogen gas.

FIG. 5 is a gas chromatograph for detecting the PTC phenylalanine aminoacid derivative prepared under the above described conditions. The gaschromatography is carried out according to the following condition.

    ______________________________________                                        Instrument (detector)                                                                          HP5890 (ECO)                                                 Carrier gas (flow rate)                                                                        helium (1.5 ml/minute)                                       Column           ULTRA #2 (crosslinked 5%                                                      phenylmethyl silicone)                                                        length 25 m, internal diameter                                                0.32 mm, film thickness 0.17 μm                           Oven temperature Initial temp. 50° C. 1 minute                                          Rinsing rate 10° C./minute                                             End temp. 300° C. 15 minutes                          Inlet opening temperature                                                                      250° C.                                               Detector temperature                                                                           300° C.                                               ______________________________________                                    

A peak (a) is obtained when 5 pmol of the sample PTC phenylalaninederivative is analyzed, another peak (b) is obtained when 500 fmol isanalyzed, and still another peak (c) is .obtained when 50 fmol isanalyzed. Based on these results, the detection limit of the presentinvention is compared with that of the conventional method utilizing thePTH derivative (PTH method). As shown in the Table 1, the detectionsensitivity of the invention is extremely high (1 pmol=1×10⁻¹² mol, 1fmol=1×10⁻¹⁵ mol).

                  TABLE 1                                                         ______________________________________                                        Detection method    Detection limit                                           ______________________________________                                        PTH method          1 pmol                                                    Inventive method    50 fmol or less                                           ______________________________________                                    

The embodiment 2 shows that reaction yield rate between ATZ derivativeand TFEA is high in the inventive highly sensitive detection method ofthe amino acid derivative. In this embodiment, a dipeptide of Phe-Gly isused. The PTC phenylalanine derivative is prepared in the same manner asthe embodiment 1. In this embodiment, the PTC phenylalanine derivativeis analyzed by using a micro-bore fast liquid chromatography (HPLC).

The used condition of HPLC is as follows.

HPLC instrument: Applied Biosystems 130A

Column: Applied Biosystems Spheri-5RP-18 (φ1.0 mm×50 mm)

Eluent: 32% acetonitrile containing 0.1% trifluoroacetic acid

Column temperature: 55° C.

Detection wave length: 254 nm

Table 2 shows a yield of the PTC phenylalanine while the quantity of ATZderivative is varied.

                  TABLE 2                                                         ______________________________________                                        Quantity of ATZ                                                               derivative        Yield                                                       (pmol)            (pmol)  (%)                                                 ______________________________________                                        100               85      85                                                  10                8.5     85                                                  1                 0.81    81                                                  0.15              0.078   78                                                  0.05              0.035   70                                                  ______________________________________                                    

As shown, even though the starting material is 50 fmol, a yield of 70%can be obtained such that the reactivity is maintained for analyzing atrace amount of sample. In the conventional method of using the PTHderivative, the reaction yield rate of the ATZ derivative is 40% at 1pmol, 10% at 100 fmol and 4% at 50 fmol.

An object of the present invention is to provide a highly sensitive anddetection of an amino acid derivative in the amino acid sequenceanalysis of protein or peptide from the N terminal.

According to a significant aspect of the present invention, in order toanalyze an amino acid sequence of protein from an amino terminal, an ATZamino acid derivative is reacted with a volatile primary aminerepresented by a general formula X-NH₂ (X denotes a hydrocarboncontaining halogen) to produce a PTC amino acid derivative. The PTCamino acid derivative is detected by a gas chromatograph provided withan ECD. By this, novel process the high yield reaction is promoted torealize highly sensitive and ready detection of the amino acidderivative. In such a reaction system, the sample is reacted with thereactive reagent composed of the primary amine having the volatilenature, which facilitates removal of an excess the reagent after thereaction. The highly sensitive detection method of the amino acidderivative according to the invention has a great industrial value.

We claim:
 1. A highly sensitive detection method of an amino acidderivative, comprising: reacting 2-anilino-5-thiazolinone amino acidderivative with a volatile primary amine represented by the generalformula X-NH₂, wherein to produce a phenylthiocarbamyl amino acidderivative, and detecting the phenylthiocarbamyl amino acid derivativeby a gas chromatograph provided with an electron capture detector.
 2. Ahighly sensitive detection method of an amino acid derivative accordingto claim 1, wherein the 2-anilino-5-thiazolinone amino acid derivativeis obtained by Edman degradation in which a protein or a peptide isreacted with a phenylisothiocyanate to produce a phenylthiocarbamylprotein or a phenylthiocarbamyl, which is thereafter reacted with anacid.